Gene therapy for respiratory disorders requires stable gene transfer to epithelial progenitor cells, efficient engraftment of these genetically modified progenitors in the respiratory epithelium and regulated transgene expression in differentiated progeny of these cells. Recently, cells have been identified in marrow that can contribute to tissue repair and regeneration in hematopoietic and non-hematopoietic tissues. In preliminary studies by our group, transgene expressing donor epithelial cells were detected in the lung following transplantation of retrovirally transduced bone marrow into murine recipients. At 2-5 months post-transplant, -1% of cytokeratin positive epithelial cells were EGFP transgene positive donor cells. Based on these data our overall hypothesis is that stem cells and their progeny can engraft in the respiratory epithelium and contribute to alveolar epithelial layer maintenance and regeneration. In studies undertaken in our currently funded grant, we are characterizing the stem cells in marrow capable of contributing to alveolar epithelium. In the proposed study, we will investigate another source of stem cells, embryonic stem cells, to differentiate into respiratory epithelium. This study has the following specific aims: 1) Maintain three HES cell lines (WA01, WA09 and UC06) in vitro in an undifferentiated state. 2) Investigate the conditions which induce HES cells to differentiate into respiratory epithelium. The technical expertise and information gained in these studies will facilitate the development of a program to study human embryonic stem cell culture and controlled respiratory differentiation. The overall goal of these studies is to further our understanding of the biology of human embryonic stem cells, and their potential to serve as vehicles for therapeutic cell delivery to the respiratory epithelium. [unreadable] [unreadable] [unreadable]